Modeling of energy migration and trapping in purple bacteria. Analysis of extreme formulae

Homogeneous pigment ensembles similar to those of purple bacteria Rhodospirillum rubrum were studied. Two formulae were advanced for the limiting values of excitation lifetime and quantum yield of excitation trapping in these ensembles, provided all reaction centers are in an active state. It was demonstrated by mathematical modeling that these limiting values strictly depend on three parameters of molecular ensembles: the numbers of core-bacteriochlorophyll molecules per reaction center, the values of rate constants for excitation trapping in reaction centers, and excitation wasteful deactivation in all molecules. The excitation lifetime and quantum yield were proved to approach their limiting values as the rate constants of excitation intermolecular migration increase. The closeness of experimental values for two above mentioned functions to their calculated limiting values proves the migration-limited type of the photosynthetic unit investigated and a high efficiency of excitation trapping in its reaction centers.     

Efficiency of excitation energy trapping in the green photosynthetic bacterium Chlorobaculum tepidum

During the millions of years of evolution, photosynthetic organisms have adapted to almost all terrestrial and aquatic habitats, although some environments are obviously more suitable for photosynthesis than others. Photosynthetic organisms living in low-light conditions require on the one hand a large light-harvesting apparatus to absorb as many photons as possible. On the other hand, the excitation trapping time scales with the size of the light-harvesting system, and the longer the distance over which the formed excitations have to be transferred, the larger the probability to lose excitations. Therefore a compromise between photon capture efficiency and excitation trapping efficiency needs to be found. Here we report results on the whole cells of the green sulfur bacterium Chlorobaculum tepidum. Its efficiency of excitation energy transfer and charge separation enables the organism to live in environments with very low illumination. Using fluorescence measurements with picosecond resolution, we estimate that despite a rather large size and complex composition of its light-harvesting apparatus, the quantum efficiency of its photochemistry is around ~87% at 20?°C, ~83% at 45?°C, and about ~81% at 77?K when part of the excitation energy is trapped by low-energy bacteriochlorophyll a molecules. The data are evaluated using target analysis, which provides further insight into the functional organization of the low-light adapted photosynthetic apparatus.     

Specific aspects of energy migration between chlorophyll molecules in the membranes of photosynthetic organisms

Several specific characteristics of energy migration in chlorophyll-protein complexes in vivo justify a suggestion of P.C. Knox to change two main equations of the theory on inductive resonance: to substitute a real lifetime of electronic excitations in donor molecules by the radiative lifetime. Critical distances for excitation migration of the main photosynthetic pigments become more stable constants; this results in appearance of more suitable formula for an average value of the mean time of intermolecular jumps of electronic excitations. In this context, the critical distances of homogenous energy migration within spectral fractions of purple bacterium, B800 and B850, were determined. The question on the possible application of Ferster’s theory to closely positioned molecules of chlorophyll and bacteriochlorophyll in vivo is also critically analyzed.     

Long-wavelength absorbing antenna pigments and heterogeneous absorption bands concentrate excitons and increase absorption cross section

The light-harvesting apparatus of photosynthetic organisms is highly optimized with respect to efficient collection of excitation energy from photons of different wavelengths and with respect to a high quantum yield of the primary photochemistry. In many cases the primary donor is not an energetic trap as it absorbs hypsochromically compared to the most red-shifted antenna pigment present (long-wavelength antenna). The possible reasons for this as well as for the spectral heterogeneity which is generally found in antenna systems is examined on a theoretical basis using the approach of thermal equilibration of the excitation energy. The calculations show that long-wavelength antenna pigments and heterogeneous absorption bands lead to a concentration of excitons and an increased effective absorption cross section. The theoretically predicted trapping times agree remarkably well with experimental data from several organisms. It is shown that the kinetics of the energy transfer from a long-wavelength antenna pigment to a hypsochromically absorbing primary donor does not represent a major kinetic limitation. The development of long-wavelength antenna and spectrally heterogeneous absorption bands means an evolutionary advantage based on the chromatic adaptation of photosynthetic organelles to spectrally filtered light caused by self-absorption.Abbreviations LHC light-harvesting complex - P primary donor - PSI Photosystem I of green plants - PS II Photosystem II of green plants - RC reaction center - X primary acceptor     

To the question of the status and the application area of the Ferster' theory to energy migration in photosynthesis

The main motive which stimulated author in writing this paper was a series of remarks by the reviewers of his articles. Some reviewers stated that Ferster' theory can not represent excitation migration in cases when electronic excitations are delocalized in several molecules. Two of reviewers even directly have proclaimed about "out of date Ferster' theory". This question is evidently of general importance. Therefore this paper contains the detailed analysis of the types of molecular ensembles and conditions which enable one to use correctly the Ferster' theory of inductive resonance.     

Chlorophyll b is involved in long-wavelength spectral properties of light-harvesting complexes LHC I and LHC II.

Chlorophyll (Chl) molecules attached to plant light-harvesting complexes (LHC) differ in their spectral behavior. While most Chl a and Chl b molecules give rise to absorption bands between 645 nm and 670 nm, some special Chls absorb at wavelengths longer than 700 nm. Among the Chl a/b-antennae of higher plants these are found exclusively in LHC I. In order to assign this special spectral property to one chlorophyll species we reconstituted LHC of both photosystem I (Lhca4) and photosystem II (Lhcb1) with carotenoids and only Chl a or Chl b and analyzed the effect on pigment binding, absorption and fluorescence properties. In both LHCs the Chl-binding sites of the omitted Chl species were occupied by the other species resulting in a constant total number of Chls in these complexes. 77-K spectroscopic measurements demonstrated that omission of Chl b in refolded Lhca4 resulted in a loss of long-wavelength absorption and 730-nm fluorescence emission. In Lhcb1 with only Chl b long-wavelength emission was preserved. These results clearly demonstrate the involvement of Chl b in establishing long-wavelength properties.     

Quantum efficiency of the minor channel for excitation migration from B800 to B875 bacteriochlorophyll fractions in purple bacteria

Excitation migration in the light-harvesting bacteriochlorophyll complexes LH1 and LH2 of purple bacteria has been studied in many experimental and theoretical works. According to the widely accepted notions, it proceeds along the descending energy stairway, B800* → 850* → 875* → P870*, where symbol * stands for excitations in the corresponding BChl fraction. In this paper we demonstrate the existence of one more way of direct excitation delivery from B800 to B875, bypassing the main route via B850. The comparative modeling enables the estimation of the mean portion of excitation that uses this minor migration way. In some real cases it may reach 9–9.5%. The values of the critical distances for excitation migration from B800 to B850 and from B800 to B875, as well as their values for arbitrary spectral shifts in BChl molecules, are determined.     

On the long-wavelength component of the light-harvesting complex of some photosynthetic bacteria

The effect of the presence of a minor antenna component in light-harvesting complexes of photosynthetic bacteria is investigated with numerical simulation employing the transition probability matrix method. A model antenna system of hexagonal symmetry is adopted, using as a working hypothesis that the minor component forms a ring around the trap. Three cases have been considered: (a) the minor component is isoenergetic with the trap, which is at lower energy than the antennas (the “supertrap”), (b) the minor component is at lower energy than the trap, which is at lower energy than the antennas (the “asymmetric gutter”), (c) the minor component is at lower energy than the trap, which is isoenergetic with the antennas (the “gutter”). It is found that the supertrap speeds up the fluorescence decay and enhances the trapping efficiency, whereas the gutter slows down the fluorescence decay and decreases the trapping efficiency. It is concluded that, in contrast to a recent suggestion (Bergström, H., R. van Grondelle, and V. Sundström. 1989. FEBS (Fed. Eur. Biochem. Soc.) Lett. 250:503-508), concentrating excitations in the vicinity of the trap by the so-called long-wavelength minor antenna component purportedly present in Rhodobacter sphaeroides and Rhodospirillum rubrum instead of improving trapping actually impedes trapping.     

Excitation transport and trapping on spectrally disordered lattices

It is widely assumed that the decay of fluorescence in photosynthetic systems can be described as a sum of exponential components and that the amplitude of each component is directly related to the absorption cross-section of the antenna pigments coupled to the fluorescing species. We present exact calculations of excited state decay in two-dimensional regular lattices of different geometries containing multiple spectral forms of antenna pigments. We illustrate by these calculations that there is no simple relation between the decay amplitudes (and resulting time-resolved excitation spectra) and the steady-state absorption spectra. Only in the limit that the electronic excitations reach a rapid equilibrium among all antenna spectral forms does the excitation spectrum depend uniquely on the spectral features of the array. Using the simulations in conjunction with our recent fluorescence studies, we examine excitation transport and trapping dynamics in photosystem I and the limitations imposed by the finite time resolution in single photon counting experiments. In particular, we show that rising components, associated with excitation transfer among different spectral forms, with lifetimes <20 ps would be undetected in a typical photon counting experiment.     

Steady-state polarized light spectroscopy of isolated Photosystem I complexes

Monomeric and trimeric Photosystem I core complexes from the cyanobacterium Synechocystis PCC 6803 and LHC-I containing Photosystem I (PS I-200) complexes from spinach have been characterized by steady-state, polarized light spectroscopy at 77 K. The absorption spectra of the monomeric and trimeric core complexes from Synechocystis were remarkably similar, except for the amplitude of a spectral component at long wavelength, which was about twice as large in the trimeric complexes. This spectral component did not contribute significantly to the CD-spectrum. The (77 K) steady-state emission spectra showed prominent peaks at 724 nm (for the Synechocystis core complexes) and at 735 nm (for PS I-200). A comparison of the excitation spectra of the main emission band and the absorption spectra suggested that a significant part of the excitations do not pass the red pigments before being trapped by P-700. Polarized fluorescence excitation spectra of the monomeric and trimeric core complexes revealed a remarkably high anisotropy (0.3) above 705 nm. This suggested one or more of the following possibilities: 1) there is one red-most pigment to which all excitations are directed, 2) there are more red-most pigments but with (almost) parallel orientations, 3) there are more red-most pigments, but they are not connected by energy transfer. The high anisotropy above 705 nm of the trimeric complexes indicated that the long-wavelength pigments on different monomers are not connected by energy transfer. In contrary to the Synechocystis core complexes, the anisotropy spectrum of the LHC I containing complexes from spinach was not constant in the region of the long-wavelength pigments, and decreased significantly below 720 nm, the wavelength where the long-wavelength pigments on the core complexes start to absorb. These results suggested that in spinach the long-wavelength pigments on core and LHC-I are connected by energy transfer and have a non-parallel average Q_y(0-0) transitions.Abbreviations PS Photosystem - P Primary donor - Chl chlorophyll - LHC light-harvesting complex - CD circular dichroism - LD linear dichroism - BisTris 2-[bis(2-hydroxyethyl)amino]-2-hydroxy-methylpropane-1,3-diol - RC reaction center     

Long-wavelength chlorophylls in photosystem I of cyanobacteria: Origin,localization, and functions

The structural organization of photosystem I (PSI) complexes in cyanobacteria and the origin of the PSI antenna long-wavelength chlorophylls and their role in energy migration, charge separation, and dissipation of excess absorbed energy are discussed. The PSI complex in cyanobacterial membranes is organized preferentially as a trimer with the core antenna enriched with long-wavelength chlorophylls. The contents of long-wavelength chlorophylls and their spectral characteristics in PSI trimers and monomers are species-specific. Chlorophyll aggregates in PSI antenna are potential candidates for the role of the long-wavelength chlorophylls. The red-most chlorophylls in PSI trimers of the cyanobacteria Arthrospira platensis and Thermosynechococcus elongatus can be formed as a result of interaction of pigments peripherally localized on different monomeric complexes within the PSI trimers. Long-wavelength chlorophylls affect weakly energy equilibration within the heterogeneous PSI antenna, but they significantly delay energy trapping by P700. When the reaction center is open, energy absorbed by long-wavelength chlorophylls migrates to P700 at physiological temperatures, causing its oxidation. When the PSI reaction center is closed, the P700 cation radical or P700 triplet state (depending on the P700 redox state and the PSI acceptor side cofactors) efficiently quench the fluorescence of the long-wavelength chlorophylls of PSI and thus protect the complex against photodestruction.     

Picosecond processes in chromatophores at various excitation intensities

The aim of this paper is to review and discuss the results obtained by fluorescence and absorption spectroscopy of bacterial chromatophores excited with picosecond pulses of varying power and intensity. It was inferred that spectral and kinetic characteristics depend essentially on the intensity, the repetition rate of the picosecond excitation pulses as well as on the optical density of the samples used. Taking the different experimental conditions properly into account, most of the discrepancies between the fluorescence and absorption measurements can be solved. At high pulse repetition rate (>10⁶ Hz), even at moderate excitation intensities (10¹⁰–10¹¹ photons/cm² per pulse), relatively long-lived triplet states start accumulating in the system. These are efficient (as compared to the reaction centers) quenchers of mobile singlet excitations due to singlet-triplet annihilation. The singlet-triplet annihilation rate constant in Rhodospirillum rubrum was determined to be equal to 10^-9 cm³ s^-1. At fluences >10¹² photons/cm² per pulse singlet-singlet annihilation must be taken into account. Furthermore, in the case of high pulse repetition rates, triplet-triplet annihilation must be considered as well. From an analysis of experimental data it was inferred that the singlet-singlet annihilation process is probably migration-limited. If this is the case, one has to conclude that the rate of excitation decay in light-harvesting antenna at low pumping intensities is limited by the efficiency of excitation trapping by the reaction center. The influence of annihilation processes on spectral changes is also discussed as is the potential of a local heating caused by annihilation processes. The manifestation of spectral inhomogeneity of light-harvesting antenna in picosecond fluorescence and absorption kinetics is analyzed.Abbreviations LHA light-harvesting antenna - RC reaction center     

To the question of Förster theory status and applicability to intermolecular energy migration in photosynthesis

The writing of this paper has been driven by a series of remarks made by reviewers of author’s works in several journals. They asserted that the Förster theory of inductive resonance is inapplicable in cases when electronic excitations are delocalized over several molecules, or plainly dismissed the theory as “out of date”. Since this is doubtlessly a question of general importance, this paper offers a detailed analysis of the types of molecular ensembles and conditions where the use of Förster theory is well founded.

     

Single molecule spectroscopy on the light-harvesting complex II of higher plants.

Spectroscopic and polarization properties of single light-harvesting complexes of higher plants (LHC-II) were studied at both room temperature and T < 5 K. Monomeric complexes emit roughly linearly polarized fluorescence light thus indicating the existence of only one emitting state. Most probably this observation is explained by efficient triplet quenching restricted to one chlorophyll a (Chl a) molecule or by rather irreversible energy transfer within the pool of Chl a molecules. LHC-II complexes in the trimeric (native) arrangement bleach in a number of steps, suggesting localization of excitations within the monomeric subunits. Interpretation of the fluorescence polarization properties of trimers requires the assumption of transition dipole moments tilted out of the symmetry plane of the complex. Low-temperature fluorescence emission of trimers is characterized by several narrow spectral lines. Even at lowest excitation intensities, we observed considerable spectral diffusion most probably due to low temperature protein dynamics. These results also indicate weak interaction between Chls belonging to different monomeric subunits within the trimer thus leading to a localization of excitations within the monomer. The experimental results demonstrate the feasibility of polarization sensitive studies on single LHC-II complexes and suggest an application for determination of the Chl transition-dipole moment orientations, a key issue in understanding the structure-function relationships.     

Spectral and functional comparisons between the carotenoids of the two antenna complexes of Rhodopseudomonas capsulata

The spectral and functional properties of carotenoids associated with each of the two light-harvesting complexes of the Rhodopseudomonas capsulata photosynthetic antenna system have been distinguished by studying mutants lacking one or the other complex. In mutants containing only the light-harvesting I complex (LH-I), the absorption spectrum of the carotenoids is blue-shifted compared to wild type. Carotenoid absorption in mutants possessing only the light-harvesing II complex (LH-II) complex is red-shifted. The circular dichroism spectrum of carotenoids in each complex is also distinctive. Although carotenoids in each complex function with approximately the same efficiency in harvesting and transmitting light energy for photosynethesis, only the carotenoids associated with LH-II undergo an electrochromic bandshift upon generation of a transmembrane potential. These observations are interpreted to indicate that both the orientation of carotenoid molecules with respect to the plane of the membrane, and the immediate electrochemical environment of these molecules differ in the two light-harvesting complexes.     

Photosynthetic activity of far-red light in green plants

We have found that long-wavelength quanta up to 780 nm support oxygen evolution from the leaves of sunflower and bean. The far-red light excitations are supporting the photochemical activity of photosystem II, as is indicated by the increased chlorophyll fluorescence in response to the reduction of the photosystem II primary electron acceptor, Q(A). The results also demonstrate that the far-red photosystem II excitations are susceptible to non-photochemical quenching, although less than the red excitations. Uphill activation energies of 9.8+/-0.5 kJ mol(-1) and 12.5+/-0.7 kJ mol(-1) have been revealed in sunflower leaves for the 716 and 740 nm illumination, respectively, from the temperature dependencies of quantum yields, comparable to the corresponding energy gaps of 8.8 and 14.3 kJ mol(-1) between the 716 and 680 nm, and the 740 and 680 nm light quanta. Similarly, the non-photochemical quenching of far-red excitations is facilitated by temperature confirming thermal activation of the far-red quanta to the photosystem II core. The observations are discussed in terms of as yet undisclosed far-red forms of chlorophyll in the photosystem II antenna, reversed (uphill) spill-over of excitation from photosystem I antenna to the photosystem II antenna, as well as absorption from thermally populated vibrational sub-levels of photosystem II chlorophylls in the ground electronic state. From these three interpretations, our analysis favours the first one, i.e., the presence in intact plant leaves of a small number of far-red chlorophylls of photosystem II. Based on analogy with the well-known far-red spectral forms in photosystem I, it is likely that some kind of strongly coupled chlorophyll dimers/aggregates are involved. The similarity of the result for sunflower and bean proves that both the extreme long-wavelength oxygen evolution and the local quantum yield maximum are general properties of the plants.     

The currently accepted model of primary energy conversion in the plant photosystems must be substantially modernized

According to the current model of primary events in plants, electronic excitations generated in antenna chlorophylls (Chl) by the light rapidly migrate within vast Chl ensembles, reach the reaction centres (RCs) and initiate the primary photoreactions of electron transfer from the RC special pairs (P700 and P680 in plant photosystems). A minor portion of electronic excitations is lost en route, in particular, via fluorescence of Chl a. A number of fluorescence parameters in vivo had been reliably established in many independent studies. Based on these parameters, the author calculated a dimensionless value, the ratio of fluorescence yields emitted by PS-2 and PS-1 Chl a ensembles. The ratio proved to differ 5-10 times from that obtained in experiments. Such a divergence seems to indicate an internal discrepancy in the currently used model. The author proposes a substantial modernization of the model by introducing a new subpicosecond state for RCs, which precedes the primary reaction of electron transfer from the RC special pairs.     

Singlet-singlet annihilation kinetics in aggregates and trimers of LHCII

Singlet-singlet annihilation experiments have been performed on trimeric and aggregated light-harvesting complex II (LHCII) using picosecond spectroscopy to study spatial equilibration times in LHCII preparations, complementing the large amount of data on spectral equilibration available in literature. The annihilation kinetics for trimers can well be described by a statistical approach, and an annihilation rate of (24 ps)(-1) is obtained. In contrast, the annihilation kinetics for aggregates can well be described by a kinetic approach over many hundreds of picoseconds, and it is shown that there is no clear distinction between inter- and intratrimer transfer of excitation energy. With this approach, an annihilation rate of (16 ps)(-1) is obtained after normalization of the annihilation rate per trimer. It is shown that the spatial equilibration in trimeric LHCII between chlorophyll a molecules occurs on a time scale that is an order of magnitude longer than in Photosystem I-core, after correcting for the different number of chlorophyll a molecules in both systems. The slow transfer in LHCII is possibly an important factor in determining excitation trapping in Photosystem II, because it contributes significantly to the overall trapping time.     

Fluorescence spectroscopy of single photosynthetic light-harvesting supramolecular systems

Recent spectroscopic studies of photosynthetic light-harvesting supramolecular complexes at the single supramolecule level are reviewed. This report describes the “single-molecule” investigation on light-harvesting complex 2 (LH2) of purple photosynthetic bacteria, phycobiliproteins of cyanobacteria and red algae, light-harvesting complex 2 (LHC2) of higher plants, and chlorosomes of green photosynthetic bacteria. Unique behaviors and spectral features of single light-harvesting apparatus have been unraveled that were hidden by the ensemble averaging of many of the complexes. The information obtained with be useful for understanding the electronic structures and energy-transfer mechanism of photosynthetic light-harvesting supramolecular systems.